Hyperglycemia is the principal factor in the genesis of diabetic complications and is also responsible for many of the metabolic derangements that occur in diabetes, but the mechanisms by which this occurs are unknown. Recent work by us and others has shown that some effects of excess glucose are mediated by products of the hexosamine pathway; the rate-limiting step in this pathway is the conversion of fructose-6-phosphate (F6P) to glucosamine-6-phosphate (GlcN6P) by the enzyme glutamine:F6P amidotransferase (GFA). For example, overexpression of GFA or treatment of cells with glucosamine induces resistance to insulin stimulation of glucose transport and glycogen synthase, and glucosamine causes insulin resistance in whole animals. We present preliminary data that overexpression of GFA in transgenic animals leads to insulin resistance and obesity. Consistent with this, in nondiabetic humans GFA activity is inversely related to in vivo glucose disposal rates and directly related to obesity. These relationships are altered in NIDDM subjects. Thus the hexosamine pathway not only mediates aspects of glucose toxicity but may also be an important regulatory of normal glucose homeostasis and may be involved in the pathogenesis of NIDDM itself. We propose to characterize the effects of alterations in GFA activity on glucose homeostasis in animals and to analyze the relationships among GFA activity, insulin resistance, and NIDDM in humans. Specifically: 1. We will analyze the insulin resistance to glucose disposal of mice overexpressing GFA in muscle and fat and explore the generality of the insulin resistance. We will measure insulin sensitivity of glucose disposal in vivo. The degree of insulin resistance will be studied in two independent lines of heterozygous and homozygous animals and correlated with the flux of glucose into UDP-GlcNAc. Insulin-stimulated glucose uptake, glycogen synthase, glycolysis, lipolysis, and lipogenesis will be measured in isolated muscle and cultured adipocytes. Attempts will be made to "cure" the animals by treatment with GFA inhibitors. 2. The mechanisms for these effects of hexosamines will be examine by studying the distal regulators of glycogen synthase (PP1 and glycogen synthase kinase) and the number and distribution of GLUT4 transporters in muscle. 3. Mice overexpressing GFA in liver exhibit obesity. We will perform in vivo measurement of glucose disposal and hepatic glucose output, and study gluconeogenic rates and insulin sensitivity in vitro in primary cultured hepatocytes. 4. The mice overexpressing GFA in liver or muscle will be cross-bred and their phenotypes analyzed as above. 5. We will continue the investigation of GFA activity and its regulation in human skeletal muscle cell cultures, comparing subjects with NIDDM and obesity to controls. We hope that these studies will lead to a better understanding of the pathogenesis of NIDDM and insulin resistance, and eventually also lead to interventional strategies for preventing some of the toxic effects of hyperglycemia in diabetes even in the absence of perfect glycemic control.